Circular dichroism (CD) spectroscopy is a spectroscopic technique where the CD of molecules is measured over a range of wavelengths. CD spectroscopy is used extensively to study chiral molecules of all types and sizes, and finds important applications in the study of large biological molecules. A primary use is in analyzing the higher order structure or conformation of macromolecules, particularly proteins. As higher order structure, for example secondary structure, is sensitive to its environment, temperature or pH, circular dichroism can be used to observe how the structure changes with environmental conditions or on interaction with other molecules. Structural, kinetic and thermodynamic information about macromolecules can be derived from circular dichroism spectroscopy.
CD calibration is required to ensure that measured CD spectra have the correct magnitude. Various factors affect measured CD magnitude, including optical imperfections in the instrument, detector non-linearity, detector polarization bias response, gains (AC and DC) in the electronic detection chain and photoelastic modulator (PEM) calibration. Ideally, CD calibration should correct for all of these error contributions across the entire wavelength range of the instrument. Calibration of CD instruments currently depends upon the use of chemical samples prepared to a prescribed concentration and measured in a cell of defined pathlength. There are several drawbacks to the use of standards such as these, including issues with accurate preparation, degradation over time, limited number or range of wavelengths over which the standard is applicable, and probably most importantly, the fact that, to qualify as a standard, the CD spectrum of the chemical sample itself has to initially be determined independently by some ‘absolute’ method. At present, no chemical CD standards exist which are traceable to a standards laboratory such as NIST or NPL.
It would be highly desirable if a solid optical CD calibration device could be developed whose theoretical CD spectrum could be computed accurately from the known configuration and optical constants of the materials comprising the device. The advantage of such an approach is that the refractive indices (including ordinary and extraordinary indices for birefringent materials) for a large number of optical materials have been determined to very high accuracy and can be modeled using well established dispersion formulae. Furthermore, materials exist, both birefringent and isotropic, which have transparency over the entire wavelength range of interest (170 nm to 1000 nm+), offering the potential for a truly wideband CD magnitude reference standard.
Several optical CD calibration devices have been described in the literature, however these suffer from certain drawbacks. For example, certain optical CD calibration devices described by the prior art are not applicable to the UV-VIS region. Moreover, a further drawback of the devices described in the prior art is that they fail to adequately take account of the effect of beam geometry (incident angle and especially divergence) on the CD signals produced. There remains a need in the art for a device that will provide more accurate calibration.
Accordingly, the present invention provides improved devices for dichroism measurements.